Project Summary/Abstract Integral membrane proteins act as critical sensors that respond to intra- and extra-cellular stimuli. These proteins are involved in many homeostatic cellular functions such as tension/mechanosensation and osmosensation, and mutations in these sensors can cause pathophysiological states. In this proposal, we will study the structure and function of the mechanosensitive ion channel, Piezo1, and the osmotic sensing volume- regulated anion channels (VRACs). Both channels were recently identified in one of the co-PI?s lab and are active targets for structural studies. Mechanically activated ion channels are thought to be responsible for hearing, sensing touch/pain, but also sensing arterial blood pressure, and lung and bladder inflation. Piezos are mechanosensitive ion channels essential for touch, proprioception, vascular biology, red blood cell morphology, and respiratory physiology. Piezo1 senses mechanical force in lipid bilayers; however, how membrane tension is sensed by these proteins and transmitted into ion channel gating is not known. Recently, we and others have solved <4 resolution structures of Piezo1, however in all structures key portions of Piezo1 were not well resolved, hindering mechanistic understanding of how mechanosensation and ion channel activity are coupled. We propose several approaches to build off our initial success and using new structures test hypotheses to address the remaining structural and mechanistic questions about Piezo1. The cellular response to osmotic pressures beyond the homeostatic range is critical for survival and yet significantly contributes to damage caused by cerebral ischemia, stroke, trauma, and hyponatremia . Cell swelling caused by hypo-osmotic stress activates ion channels including volume-regulated anion channels (VRAC). VRAC is a diverse set of heteromeric channels of undefined complexity composed of the essential LRRC8A (?SWELL1?) subunit and any of 4 other LRRC8 family members. Despite recent high-resolution structures of homo-hexameric LRRC8A from our group and others, the number of subunits, exact composition and stoichiometry of VRAC are still unknown. Heterologous expression has revealed that important differential physiological functions of VRAC are dependent on the identity of associating subunits. The primary focus of our proposed studies is the elucidation of the structure and subunit arrangement of VRACs using high-resolution cryo-electron microscopy (cryo-EM), and how each of the various assemblies accomplishes different functions. We believe this proposal targeting these important ion channels will significantly impact our knowledge of cell volume homeostasis in response to environmental stresses, as well as cell response to membrane tension, impinging on all vertebrate organ systems since Piezos and VRACs are nearly ubiquitous.